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Refined
fermentation process development strategies have geared up
microbes as a sustained source of future drugs. Dr H
Sivaramkrishna, President and Dr Girish Mahajan, Group Head,
Anti-infective Screening & Prokaryote Isolation, Department of
Natural Products, Taj Pharmaceuticals Limited illustrate the
pre-eminence fo microbes in the pharma industry focusing on three
prominent areas: anti-microbial (anti-bacterial and anti-fungus),
anti-cancer and anti-diabetic drugs.
Today, microbiology is a major participant in the global
pharmaceutical and neutraceutical industry. The microbes keep on
producing novel metabolites as they move into the diverse
ecological units. From the biologically active compounds that have
been obtained so far from microbes, 45 percent are produced by
actinomycetes, 38 percent by fungi and 17 percent by unicellular
bacteria. However, the development of resistance in microbes and
tumour cells has become a major problem and requires much research
effort to combat it. Microbes have always been a better resource
for getting lead molecule with novel scaffold to overcome any such
limitation of existing drugs.
Mini medicine making factories
The versatility of microbial drugs is gigantic. Secondary
metabolites of microbes are exceedingly important to our health
and nutrition. These metabolites have tremendous economic
importance. Per se the antiinfective market amounts to almost
$62.5 billion and includes about 166 anti biotics and derivatives
such as the -lactam peptide ant ibiotics, the macrolide polyketide
erythromycin, tetracyclines, aminoglycosides, daptomycin, and
others. Additional rudimentary pharma products produced by microrganisms are anti diabetic drugs, hypocholesterolemic agents,
enzyme inhibitors, immunosuppressants, herbicides, antihelmintics,
biopesticides and anti tumour compounds, some having markets of
over $1 billion per year (Table 1). Microbial products comprising
of actinomycetes, fungi and myxobacteria continued to play a
highly significant role in the drug discovery and development
process. The power of the microbial culture in the competitive
world of commercial synthesis can be appreciated by the fact that
even simple molecules (ie L-glutamic acid and L-lysine), are made
by fermentation rather than by chemical synthesis. Most natural
products are made by fermentation technology. Owing to technical
improvements in screening programmes, and separation and isolation
techniques, the number of natural compounds discovered exceeds one
million by end of 2005. Of these five percent have a microbial
origin. Of all the reported natural products, approximately 20-25
percent show biological activity, and of these, approximately 10
percent have been obtained from microbes. Furthermore, from these
biologically active compounds, which have been obtained so far
from microbes, 45 percent are produced by actinomycetes, 38
percent by fungi and 17 percent by unicellular bacteria.
Although, microbes are remarkably superior in presenting us with
an amazing array of precious drugs, wild strains usually produce
them in low amount, usually few micrograms to milligrams per litre
of culture broth. The fermentation microbiologist, however,
desires an 'extravagant' strain, which will overproduce and
excrete the desired compound that can be isolated and marketed.
During the screening stage, the microbiologist searches for
microbes with weak regulatory mechanisms. Once a desired strain is
found, a strain improvement programme is taken up to improve
yields of desired products by modification of culture conditions,
mutation and other molecular biology techniques. Thousand-fold
increases have been recorded for small metabolites. Such refined
fermentation process development strategies geared up microbes as
sustained source of future drugs. To illustrate the preeminence of
microbes in pharma industry we have focused on three prominent
areas anti-microbial (anti-bacterial and antifungals), anti-cancer
and anti-diabetic drugs.
Anti-microbial drugs
The eon of the drugs from microbes began in 1928, when Alexander
Fleming discovered in a petri-plate seeded with a bacteria named
Staphylococcus aureus, that a compound produced by a mould killed
the bacteria. "When I woke up just after dawn on September 28,
1928, I certainly didn't plan to revolutionise all medicine by
discovering the world's first antibiotic, or bacteria killer,"
Fleming would later say, "But I guess that was exactly what I
did." Ernst Chain and Howard Florey became interested in his work,
and is olated, characterised active compound from it. The mould,
classified as Penicillium notatum, produced an active agent that
was named penicillin. By history gramicidin, the first clinically
tested antibiotic, was the first natural antibiotic discovered
through a deliberate, systematic search for antibacterial
compounds by René Dubos. This discovery helped revive the stalled
interest in penicillin and launched the era of antibiotics. Later,
penicillin was used as a potent antibacterial compound during
World War II. Streptomycin, the first antibiotic remedy for
tuberculosis, was first isolated from an actinobacterium
Streptomyces griseus in 1943 by Albert Schatz, a graduate student,
in the laboratory of Selman Abraham Waksman at Rutgers University.
Chloramphenicol, a broad spectrum antibiotic was originally
isolated from the soil microbe Streptomyces venezuelae in 1947.
The tetracyclines are a large family of antibiotics produced my
many species of streptomyces, which were discovered as natural
products by Benjamin Minge Duggar and first described in 1948.
Tetracycline was then discovered by Lloyd Conover in the research
departments of Pfizer13,14.
Many small molecule drugs were approved in the antibacterial area
from the beginning of 2003 to 2008. These included daptomycin
which was launched as Cubicin by Cubist Pharmaceuticals in 2003,
which was produced by Streptomyces roseosporus. It was the first
of a new class of cyclic lipopeptides. It has been approved by FDA
for the treatment of infections caused by MRSA and other Gram
positive pathogens15. Wyeth had their modified tetracycline
derivative, tigecycline, approved as a drug designed to overcome
the tetracycline resistance pump in pathogenic bacteria. Recently
launched Doripenem (Doribax, Johnson & Johnson) is a distant
analogue of a carbapenem called theinamycin. The latter was
produced by Streptomyces cattleya. Ceftobiprole (BAL5788) is the
first, broad-spectrum, anti-MRSA fourth generation cephalosporin
antibiotic with activity against a range of difficult-to-treat
gram-positive and gram-negative hospital and community-acquired
pathogens including methicillin-resistant S aureus (MRSA) and
Pseudomonas aeruginosa16. In 2008 Ceftobiprole was licensed from
and co-developed with Basilea Pharmaceutica. Janssen-Cilag will
market ceftobiprole in Switzerland under the trade name Zevtera.
Fungal infections range from superficial conditions of the skin (e.g
ringworm and athlete's foot) and nails (onychomycoses) to
disseminated life threatening diseases. Serious invasive fungal
infections caused by Candida spp, Cryptococcus neoformans,
Aspergillus spp, Pneumocystis carinii and Histoplasma capsulatum,
represent an increasing threat to human health. The prevalence of
these systemic fungal infections has increased significantly
during the past two decades. For nearly 30 years since 1955,
amphotericin B, a polyene antibiotic (produced by Streptomyces
nodosus), was the sole drug available to control serious fungal
infections. To overcome its nephrotoxicity and solubility issues
it has been marketed in different formulation brands such as
Amphotec, Abelcet, Ambisome, fungisome, fungisome etc. Even today
in the crowd of new antifungals this microbial product has unique
market position. In the past decade in the antifungal area, of the
five drugs approved, four were azoles and the semisynthetic
echinocandin derivative, anidulofungin (microbial derived), which
was approved for use in the US in early 2006. Micafungin (trade
name Mycamine) is another echinocandin antifungal drug developed
by Astellas Pharma and gained approval in the European Union in
April, 2008. Discovery of echinocandins stemmed from studies on
papulacandins isolated from a fungal strain of Papularia
sphaerosperma.
Thus microbes have remained as consistent sources for new
antimicrobial agents overcoming new snags and challenges.
Anti-cancer drugs
The second area where microbial drugs are very successful in
making their unremitting influence is as cancer chemotherapeutic
agents. The discovery of actinomycin has led to venture into
microbial world in the quest for anticancer compounds. Among the
approved products deserving special attention are actinomycin D,
anthracyclines (daunorubicin, doxorubicin, epirubicin, pirirubicin
and valrubicin), bleomycin, mitosanes (mitomycin C), anthracenones
(mithramycin, streptozotocin and pentostatin), enediynes (calcheamycin),
taxol and epothilones. Actinomycin A, was the first antibiotic
isolated from actinomycetes, Actinomyces antibioticus (now
Streptomyces antibioticus) by Waksman and
 Woodruff. Despite the
toxicity, however, it has served well against Wilms tumour in
children. The anthracyclines are some of the most effective
antitumour compounds developed, and are effective against more
types of cancer than any other class of chemotherapy agents. They
are used to treat a wide range of cancers, including leukaemia,
lymphomas, and breast, uterine, ovarian and lung cancers. Their
main adverse effects are heart damage (cardiotoxicity), which
considerably limits their usefulness, and vomiting.
The first anthracycline discovered was daunorubicin (daunomycin)
in 1966, which is produced naturally by Streptomyces peucetius.
Doxorubicin (adriamycin) was developed in 1967. Another
anthracycline, epirubicin approved by the FDA in 1999, is favoured
over doxorubicin in some chemotherapy regimens as it appears to
cause fewer side effects. Epirubicin is primarily used against
breast and ovarian cancer, gastric cancer, lung cancer and
lymphomas. Valrubicin is a semisynthetic analog of doxorubicin
approved as a chemotherapeutic drug in 1999 and is used to treat
bladder cancer. Bleomycin is a non-ribosomal glycopeptide
microbial metabolite produced as a family of structurally related
compounds by the bacterium Streptomyces verticillus. It was first
reported by Umezawa et al19 in 1966, and FDA approved it in 1973.
Mitosanes are composed of several mitomycins that are formed
during the fermentation of Streptomyces caespitosus. Although the
mitosanes are excellent antitumour agents, they have limited
utility owing to their toxicity. Mitomycin C was approved by the
FDA in 1974, showing activity against several types of cancer
(lung, breast, bladder, anal, colorectal, head and neck),
including melanomas and gastric or pancreatic neoplasms20.
Mithramycin (plicamycin) is an antitumour cum antibacterial
aromatic polyketide produced by Streptomyces argillaceous. It is
one of the classical chemotherapy drugs used in the treatment of
testicular cancer, disseminated neoplasms and hypocalcaemia.
Streptozotocin, a glucosamine-nitroso-urea, is a microbial
metabolite with antitumour properties. It is produced by
Streptomyces achromogenes. It is toxic to cells by causing damage
to DNA, although other mechanisms may also contribute. The
compound is selectively toxic to the ß-cells of the pancreatic
islets. In 1982, FDA granted approval for streptozotocin as a
treatment for pancreatic islet cell cancer.
Pentostatin (deoxycoformycin), a purine analogue is an anticancer
drug produced by S antibioticus. It interferes with the cell's
ability to process DNA.
The FDA granted approval for pentostatin in 1993. Calicheamicins
are highly potent antitumour microbial metabolites of the enediyne
family produced by Micromonospora echinospora. Their antitumour
activity is apparently due to the cleavage of double-stranded DNA.
They are highly toxic, but it was possible to introduce one such
compound into the clinic by attaching it to an antibody that
delivered it to certain cancer types selectively. This ingenious
idea of the Wyeth Laboratories avoided the side effects of
calicheamicin. It was approved by the FDA for use in patients over
the age of 60 years with relapsed AML who are not considered
candidates for standard chemotherapy.
Taxol (paclitaxel) is reported to be produced by the endophytic
fungi Taxomyces andreanae and Nodulisporium sylviforme. This
compound inhibits rapidly dividing mammalian cancer cells by
promoting tubulin polymerisation and interfering with normal
microtubule breakdown during cell division. In 1992, taxol was
approved for refractory ovarian cancer, and today it is used
against breast and advanced forms of Kaposi's sarcoma. Taxol sales
amounted to $1.6 billion in 2006 for Bristol Myers-Squibb,
representing 10 percent of the company's pharma sales and its
third largest selling product. The epothilones are macrolides
originally isolated from a myxobacterium, Sorangium cellulosum.
This microbial product fuelled the hunt for novel class of
microbes for drugs discovery. They have mode of action similar to
taxol. Moreover, they are generally five to 25 times more potent
than taxol in inhibiting cell growth in cultures. Five analogs are
now undergoing investigation as candidate anticancer drugs, and
their preclinical studies have indicated a broad spectrum of
antitumour activity, including taxol-resistant tumour cells. One
epothilone, ixabepilone, was approved in October 2007 by the FDA
for use in the treatment of aggressive metastatic or locally
advanced breast cancer no longer responding to currently available
chemotherapies. The discovery of Salinosporamide A (NPI-0052)
showed that the best has yet to come in cancer area. It is a novel
anticancer compound from marine actinomycetes, Salinispora tropica.
Salinosporamide A is a potent proteasome inhibitor used as an
anticancer agent and it had recently entered phase I human
clinical trials for the treatment of multiple myeloma only three
years after its discovery.
Anti-diabetic drugs
Actinomycetes are reported to produce potent anti-diabetic
molecules, which target various glucosidase enzymes. Acarbose
(produced by Actinoplanes strain SE 50) is an oral alpha-glucosidase
and alpha-amylase inhibitor that was first launched by Bayer in
Switzerland in 1989 for the oral treatment of type II diabetes
mellitus. It is currently marketed in various countries worldwide,
including the US, the UK, Canada, France, Germany, Italy and
Japan. After intensive clinical development, acarbose (brand name
Glucobay) was introduced into the market in Germany in 1990 for
the treatment of diabetes and has since been successfully marketed
in Europe and Latin America. In 1996, acarbose was introduced in
the US under the brand name Precose.
Voglibose is an alpha-glucosidase inhibitor used for lowering
post-prandial blood glucose levels in people with diabetes
mellitus. It is produced and marketed in India by trade name Volix
(Ranbaxy Labs) and Vocarb (Glenmark). It is produced by the
actinomycete species, Stretomyces hygroscopicus subspecies
limoneus.
Valielamine, a precursor of Voglibose and a new aminocyclitol has
been isolated from the fermentation broth of Streptomyces
hygroscopicus subspecies limoneus. It has more potent a-glucosidase
inhibitory activity against porcine intestinal sucrase, maltase
and isomaltase than valienamine, validamine and hydroxyvalidamine
which were reported as building blocks of validamycins and
microbial oligosaccharide a-glucosidase inhibitors. It is under
preclinical development at Sikaqaku, Tokyo, Japan.
Tendamistat (produced by Streptomyces tendae 4158 and Streptomyces
lividans) is an extracellular polypeptide containing 74 amino
acids, shows significant biological activity as an a-amylase
inhibitor.and has shown prominent application in treatment of
diabetes mellitus. Due to its resistance against most hydrolytic
enzymes, Tendamistat would be orally available for diabetes
mellitus treatment. However, its immunogeneity could prevent its
further developement. It is still under development at
sanofi-aventis, Paris. Nojirimycin (produced by several strains of
Bacillus, Streptomyces), a potent inhibitor of both a- and ß-glucosidases
of different origins, Adiposin-1 (isolated from Streptomyces
calvus), inhibitor of human a-amylase are few more examples of
potential anti-diabetic compounds from microbes, with many more
still at discovery stage.
In addition to being the source for such number of new molecules
which are effective in different therapeutic areas, recent studies
have shown that soil bacteria are so friendly to human being that
, they alter the behaviour in a way similar to the effect of
anti-depressants. A study published in Neuroscience in 2007, says
levels of serotonin in brain were boosted by bacteria just as
anti-depressants.
The representative data in three major therapeutic areas
unanimously suggests that microbes form an importunate resource
for discovering and developing new drugs that address the unmet
medical needs of the masses. The concept has been exemplified by
the recent discovery of a novel potent anti gram positive lead
PM181104 by Taj Pharmaceuticals which has been isolated from an
actinobacterium obtained from marine sponge34. PM181104 is under
late toxicological studies at PLSL. Though one of the youngest
research organisation, PLSL maintains the largest collection of
diverse industrially important microbes (about 44,000 strains) and
library of their extracts (about 43,000 extracts), and a
state-of-the-art facility to develop drugs from microbes. Many
promising leads from microbes and their semi-synthetic derivatives
are in pre-clinical pipeline in anti-infective, anti-cancer,
anti-diabetes and anti-inflammation area.
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